Transgene expression in erythroid colonies.

ES cells containing the targeted −4.0 kb transgene were differentiated in primary culture for 5 days. Then, embryoid bodies (EB) were disrupted to single cells and plated in a secondary methylcellulose culture. Between 4 and 8 days of secondary culture, colonies of erythroid cells were visible as foci of red cells (globinized areas indicated with red arrows) with nonglobinized cells (black arrows). The erythroid colonies were surrounded by nonerythroid cells of unknown lineage. Original magnification, × 200. (A) Erythroid colonies were observed using either visible, (B) porphyrin-specific, or (C) EGFP-specific wavelengths. (Yellow areas indicate emission filter bleed-through of porphyrin fluorescence combined with green fluorescence) (D) To observe areas of fluorescence in the erythroid colony, the porphyrin fluorescence image was overlaid with the visible image, or (E) the EGFP fluorescence image was overlaid with the visible image. (F) To identify areas of both porphyrin and EGFP expression in the erythroid, the images from porphyrin fluorescence and EGFP fluorescence were overlaid.